Rapid analysis of amatoxins in human urine by means of affinity column chromatography and liquid chromatography-high-resolution tandem mass spectrometry

Analysis of amatoxins is of great importance as these cyclic peptides contribute to a high number of fatalities each year. Development of analytical approaches needs to focus on rapid, sensitive, and reliable methods. By establishing an affinity column chromatography-based assay using the monoclonal amanitin antibody AMA9G3 and liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) for the trace detection of α-, β-, and γ-amanitin in human urine samples to confirm ingestion, we report the first approach that extents the current status of amatoxin analysis. The presented procedure allows detection of amatoxins in human urine down to 1 ng/mL. The method was successfully validated qualitatively for α- and γ-amanitin according to international recommendations. A proof of concept was performed by analyzing 37 urine samples after suspected amatoxin consumption submitted for regular clinical toxicological analysis. Using this antibody-based enrichment strategy, acute amatoxin intoxications can be determined within 90 min and due to the high sensitivity and selectivity, a comparable approach using target specific antibodies may also be used for other toxicological relevant peptides.


Data handling and method validation
Statistical analysis was performed using Microsoft Excel 2010 (Redmond, WA, USA).
ACD/Chem Sketch freeware version 2015 was used for the calculation of the exact masses of the amatoxins as well as the internal standard (IS) and TF Xcalibur Qual Browser version 4.1 for qualitative data evaluation.Qual Browser settings were as follows: detector type, MS; peak algorithm, genesis; plot type, mass range; m/z of each substance; mass tolerance, 5 ppm; mass precision, 4 decimals; smoothing, not enabled.Extracted ion chromatograms of the analytes were edited using CorelDraw X7 Version 17.0.0.491 (Munich, Germany).Method validation for α-and γ-amanitin covering selected parameters was performed according to international recommendations [1][2][3].Selectivity testing was performed using urine samples from six different donors which were extracted, analyzed, and evaluated for interferences.For carryover testing, eight extracted blank urine samples were injected after a urine sample spiked with 50 ng/mL of α-and γ-amanitin.Matrix effects (ME) and recoveries (RE) were determined according to Matuszewski et al. using three different sample sets (n = 6, concentration of α-and γ-amanitin, 10 ng/mL) [4].The first sample set (neat standard) was prepared in aqueous glycine buffer (100 mM, pH 1.8).Blank urine was spiked with α-and γ-amanitin before affinity column chromatography (sample set 3) or after affinity column chromatography (sample set 2).For αand γ-amanitin, ME were calculated using the ratio of the peak area in the presence of matrix (sample set 2) and the peak area in absence of matrix (sample set 1). Division of mean peak areas of sample set 2 by those of sample set 1 were used for calculation of the ME.Coefficients of variation (CV) for α-and γ-amanitin should not be greater than 15%.Division of mean peak areas of sample set 3 by those of sample set 2 was used for calculation of the RE.Stability of the stock and working solutions of α-and γ-amanitin as well as the IS was tested over a period of six weeks in purified water (n = 3, concentration of α-and γ-amanitin and IS, 500 ng/mL).
CVs not greater than ±15% were defined to be acceptable.Autosampler stability of processed samples was investigated over a period of four days (extracts stored at 20 °C) next to short-term stability (spiked urine samples stored 24 at 4 °C), benchtop stability (spiked urine samples stored 24 h at 22 °C), and long-term stability (spiked urine samples stored four weeks at -20 °C).One freeze and thaw cycle was conducted for the freeze and thaw stability (spiked urine samples stored for 24 h at -20 °C).Urine samples spiked with α-and γ-amanitin (final plasma concentration, 10 ng/mL, n = 3) were extracted and analyzed immediately after preparation (t0) and after the appropriate storage condition (t1).Peak area deviations as well as CVs not greater than ±15% were defined to be acceptable.

Fig. S2
Fig. S2 Reconstructed ion chromatograms obtained after analysis of an authentic urine sample used for the selectivity study

Table S1
Additional drugs included in the selectivity study.